The broad, long-term objectives of this project are to gain novel knowledge about a highly specialized calcium (Ca2+) channel - the inositol (1,4,5)-trisphosphate receptor (InsP3R). InsP3R supports a major pathway for Ca2+ release from intracellular Ca2+ stores and plays a key role in the intracellular Ca2+ signaling process. Cytosolic Ca2+ influences most fundamental cellular processes and derangement of intracellular Ca2+ signaling has been implicated in a variety of pathological conditions, such as ischemia, manic and spreading depression, hypertension, atherosclerosis, immunosuppression, cancer, and Lowe's oculocerebrorenal syndrome. Thus, the proposed studies of InsP3R are fundamental to understanding Ca2+ signaling in cells and may provide novel information relevant to multiple human diseases. The specific aims of the proposal are: 1. To identify structural determinants of InsP3R conductance. Mutations will be introduced in specific sites within the putative pore-forming region of InsP3R and properties of heterologously expressed mutant channels will be evaluated in planar lipid bilayers. 2. To elucidate the mechanism for InsP3R activation by InsP3. Mutations will be introduced in specific sites within the InsP3-binding domain region and the properties of recombinant mutant InsP3R will be evaluated by radiolabeled InsP3 binding and planar lipid bilayer assays. 3. To characterize structural determinants of InsP3R modulation. Mutations will be introduced in putative calmodulin, ATP and Ca2+ binding sites and the properties or recombinant mutant channels will be analyzed in planar lipid bilayers. 4. To evaluate a hypothesis of direct InsP3R-PIP2 (phosphatidylinositol 4,5-bisphosphate) functional coupling. Preliminary experiments presented here suggest the existence of a novel signaling InsP3R-PIP2 complex. Binding of a radiolabeled water-soluble PIP-2 analog to recombinant InsP3R fragment will be used to identify the location of a specific PIP2-binding site in the InsP3R sequence. Activity of InsP3R will be correlated with cellular PIP2 content to obtain information regarding InsP3R-PIP2 interaction in situ.